Antiarrhythmic effects of polyunsaturated fatty acids following dietary incorporation into cardiac cell membranes have been observed in recent years. The mechanisms of action are yet to be defined. In this project the effect of membrane-free polyunsaturated fatty acids delivered to isolated adult rat cardiac myocytes was investigated (in vivo these can be released from cardiac cell membranes following phospholipase action). The present study investigates the effects of DHA and arachidonic acid (AA;C20:4,n-6) o L-type calcium and K+ channel conductance in whole cell voltage-clamp experiments and on cytosolic free calcium fluorescence and contraction in adult rat indo-1 loaded cardiac myocytes. Nitrendipine (10 nM) reduced pea ICa, measured by whole cell clamp from -40 to -5 mV, twitch contraction amplitude and associated cytosolic indo-1 Ca2+ fluorescence measured during electrical stimulation (0.5Hz). DHA (5fM) abolished these effects but AA di not block the nitrendipine effects. Experiments with 10nM BAYK8644 resulted in increased twitch contraction and related cytosolic calcium which could b prevented by DHA but not AA (Fig2). DHA or AA alone had no effect. Neither DHA nor AA altered isoproterenol (1, 0.5, 0.1fM) induced increases in ICa o twitch amplitude. That DHA abolishes nitrendipine or BAYK8644 effects but has no effect alone, suggests that it binds to Ca2+ channels near dihydropyridine binding sites and interferes with ICa modulation by dihydropyridines. While DHA has no effect on inward rectifier current (Ik1 and delayed rectifier current (Ik) it accelerates the inactivation of transient outward K+ current (Ito) and decreases its magnitude. These results suggest that selective blockade of Ito and also binding to the dihydropyridine receptor in heart cells by omega-3 PUFA may protect agonist Ca2+ overload and ischemia induced arrhythmias. This action may be involved in the antiarrhythmic effects of fish oil diet in vivo both in animal models and in humans with coronary artery disease.